1. Technical Field
The present invention provides for methods of identifying compounds for treating medical conditions related to the inappropriate production of mucin, such as Pseudomonas infections of cystic fibrosis patients, bronchial pneumonia, chronic bronchitis and bronchial asthma.
2. Background
Mucins are a family of glycoproteins secreted from epithelial cells at many body surfaces, including the eyes, pancreatic ducts, gallbladder, prostate and mainly, respiratory, gastrointestinal and female reproductive tracts. Mucins are responsible for the viscoelastic properties of mucus. In the airways, mucin interacts with cilia to trap and clear pathogens and irritants. Bacterial infection of the airway epithelium is often accompanied by mucin overproduction. In addition, airway diseases such as chronic bronchitis, cystic fibrosis and asthma are characterized by mucus hypersecretion. Hypersecretion can overwhelm the ability of the cilia to finction properly. Mucociliary impairment leads to airway mucus plugging which promotes chronic infection, airflow obstruction, and sometimes death.
Nine mucin genes are known to be expressed in man: MUC 1, MUC 2, MUC 3, MUC 4, MUC 5AC, MUC 5B, MUC 6, MUC 7 and MUC 8 (Bobek, et al. (1993) J. Biol. Chem. 268:20563-9; Dusseyn, et al., (1997), J. Biol. Chem. 272:3168-78; Gendler, et al. (1991) Am. Rev.Resp. Dis. 144:S42-S47; Gum, et al. (1989) J. Biol. Chem. 264:6480-6487; Gum, et al. (1990) Biochemical and Biophysical Research Communications 171:407-415; Lesuffleur, et al. (1995) J. Biol. Chem. 270:13665-13 673; Meerzaman, et al. (1994) J. Biol Chem. 269:12932-1293 9; Porchet, et al. (1991) Biochem Biophys. Res. Comm. 175(2):414-422; Shankar, et al. (1994) Biochem. J. 300:295-298; Toribara, et al. (1997) J. Biol. Chem. 272:16398-403). Cysteine-rich domains are considered to be typical of mucin sequences, having been reported in many mucins including MUC 2 (Gum, et al. (1992), J. Biol. Chem. 267:21375-21383; Gum, et al. (1994), J. Biol. Chem. 269:2440-2446), MUC 5AC (Meerzaman, et al. (1994), J. Biol. Chem. 269:12932-12939), MUC 5B (Desseyn, et al. (1997) J. Biol. Chem. 272:3168-3178) and MUC 6 (Toribara, et al. (1997) J. Biol. Chem. 272:16398-16403) as well as in rat (Ohmori, et al. (1994) J. Biol. Chem. 269:17833-17840), pig (Eckhardt, et al. (1991) The Journal of Biological Chemistry, 266(15):9678-9686), cow (Bhargava, et al. (1990) Proc. Nat. Acad Sci. U. S. A. 97:6798-6802) and frog (Probst, et al. (1990) Biochemistry 29:6240-6244) mucins. The cysteine-rich domains in mucins show varying degrees of similarity to the D-domains of von Willebrand factor (vWF).
Cystic fibrosis (xe2x80x9cCFxe2x80x9d) commonly occurs among Caucasians (approximately 1 in 2,000 newborns). The mode of inheritance is autosomal recessive and about 5% of the normal population carries the defective gene. Affected individuals can generally live with reasonable lung function until the onset of a chronic bacterial infection. Almost all patients contract either Pseudomonas aeruginosa or Staphylococcus aureus infections. Mucus overproduction resulting from the bacterial infection damages lung function directly by plugging airways and indirectly by shielding the bacteria from endogenous and exogenous antibacterial agents. This creates a xe2x80x9cwound that does not healxe2x80x9d and causes chronic influx of inflammatory cells whose proteases degrade gas exchange tissue. Respiratory finction declines relentlessly until death results.
Current treatments fail to effect the complete eradication or prevention of these bacterial infections in cystic fibrosis patients nor do they ameliorate the overproduction of mucus. In addition, antimicrobial therapy using antibiotic therapeutic protocols have complications. Patients with CF dispose of antimicrobial agents more rapidly than do non-CF individuals, which results in the use of higher doses than those normally recommended. Strains of Pseudomonas aeruginosa (xe2x80x9cPAxe2x80x9d) can dissociate into multiple serotypic forms, which often have different antimicrobial susceptibility patterns. Since PA infection is chronic and the infecting strains of Pseudomonas aeruginosa are rarely eradicated, resistance to multiple antimicrobial agents frequently develops, thwarting antibiotic therapies. Moreover, therapeutic levels of antimicrobial agents in sputum are difficult to achieve because of poor penetration and inactivation. Mucoid exopolysaccharides of mucoid PA strains additionally present a barrier to penetration of some antibiotics. Finally, allergy to certain antibiotics (such as betalactam) precludes antibiotic therapy with some patients. Thus, as it is virtually impossible to eradicate the bacteria, it is important to find alternate therapies to improve lung function and prolong life. The ability to control mucin production may provide an alternative route to prevent or alleviate airway plugging.
In addition to its role in exacerbating pulmonary infections in cystic fibrosis patients, mucin overproduction is also a debilitating feature of chronic bronchitis, bronchial pneumonia and chronic asthma. Smoking is the most important risk factor for chronic bronchitis. Individuals dying in status asthmaticus are always observed to have mucus-obstructed airways.
Consequently, there is a need to provide therapies for reducing mucus production in individuals suffering from airway diseases such as cystic fibrosis, chronic bronchitis, bronchial pneumonia and asthma.
Relevant Literature
Recent work has suggested that MUC 4 and MUC 5AC are the most highly expressed mucins, in the upper airway (Gendler, et al. (1996) Pediatric Pulmonology 13S:290 Abstracts). Mucus secretions in the airway are produced from two different secretory cell populations, surface epithelial goblet cells and the mucous cells in the submucosal glands. MUC 5AC has been reported to be expressed primarily in the goblet cells (Hovenberg, et al. (1996) Biochem. J. 318:319-24).
Increased expression of some members of the mucin family in response to certain effectors has been reported. Recent reports have indicated that MUC 2, MUC 4 and MUC 5B are expressed at a higher level in the airways of cystic fibrosis patients compared with non-CF patients (Gendler et al. (1996) Pediatric Pulmonology 13S:290 Abstract); Li, et al. (1997) Proc. Nat. Acad Sci. (USA) 94:967-972). Steiger et al. (1995) (Am. J Resp. Cell and Molec. Biol. 12:307-314) have reported that bacterial endotoxin stimulates both the storage and release of a mucin-like molecule in airway epithelial cells. Levine, et al. (1995) (Am. J Resp. Dis. 12:196-204) have reported an increase in the steady state levels of MUC 2 MRNA in NCI-H292 cells by exposure to tumor necrosis factor-xcex1. Li et al. (Proc. Nati Acad Sci. (1997) 94:967-972) have reported that PA lipopolysaccharide (xe2x80x9cLPSxe2x80x9d) can increase transcription of the MUC 2 gene in epithelial cells. Pre-incubation of the cells with the protein tyrosine kinase inhibitor Genistein abolished the increase in transcription. Recent work in the laboratory of some of the present inventors has suggested that MUC 5AC transcription is also increased after exposure to various Gram-negative and Gram-positive bacteria in both bronchial explants and cultured airway epithelial cells. Borchers and Leikauf have recently reported (Am. J Respir. Critical Care Med. 155:A778 (1997)) that exposure of rats to acrolein, a low molecular weight aldehyde found in tobacco smoke, results in an increase in MUC-2 mRNA and concurrent secretion of mucin in airway tissue.
Partial MUC 5AC cDNA sequences have been reported (Meerzaman, et al. (1994) J. Biol. Chem., 269:12932-12939); Guyonnet-Duperat, et al. (1995) Biochem. J. 211-219). The nucleotide sequence of human gastric mucin cDNA, HGM-1, from nucleotides, 1942-2281 is 99% identical to the MUC 5AC clone JUL 32 (Guyonnet-Duperat, et al. (1995) Biochem J. 211-219) and the sequence from nucleotides 2190-2541 is 92% identical to the 5xe2x80x2 end of MUC 5AC clone NP3a (Klomp, et al. (1995) Biochem. J., 308:831-80). The sequence of the MUC 2 gene promoter has been reported (Velcich, et al. (1997) J. Biol. Chem. 272:7968-7976 (1997).
The present invention provides methods and compositions for the selection of inhibitors of mucin production. The present inventors have identified for the first time the-regulatory elements of the human MUC 5AC gene which control the response of the gene to effectors which induce the expression of MUC 5AC. Nucleotide sequence of the regulatory region is provided herein. By using these regulatory elements, particularly in combination with a readily detectable reporter gene, it is possible to identify compounds that inhibit the induction of mucin genes, particularly MUC 5AC, by various effectors, or mucomones. Inhibitor compounds thus identified are useful in therapeutic methods for the treatment of diseases and conditions associated with the inappropriate production of mucin.
The present inventors have additionally discovered that tobacco smoke, in the form of smoke-conditioned culture medium, can serve as a mucomone to induce the transcription of mucin genes, in particular, the MUC 5AC gene. Nucleotide sequences important for providing induction of MUC 5AC by tobacco smoke are identified.
The present invention thus provides polynucleotide molecules comprising one or more MUC 5AC mucomone response elements, mucomone-inducible reporter gene constructs comprising the MUC 5AC mucomone response elements operably linked to a reporter gene, and cells comprising the reporter gene constructs.
The present invention additionally provides methods for identifying compounds that inhibit mucin production using the polynucleotide molecules, reporter gene constructs and cells of the present invention. In one embodiment, the method of the present invention comprises contacting cells comprising a mucomone-inducible reporter gene construct with a test compound, contacting said cells with a mucomone, and determining the difference in reporter gene activity in response to mucomone in the presence and absence of the test compound. The method is particularly usefuil for identifying compounds that inhibit tobacco smoke-induced mucin production.
Mucomone-induced mucin production can be affected in a variety of ways, for example, by inhibiting binding of mucomones to an epithelial cell or by inhibiting any crucial step in the signal transduction cascade between binding of a mucomone and mucin gene activation, including inhibiting transcription factors that interact with mucin promoters. Such inhibitors can be used in treatments of medical conditions related to the inappropriate expression of mucin, such as, for example, cystic fibrosis, chronic bronchitis, bronchial pneumonia and bronchial asthma. The invention includes methods and compositions related to drug discovery and therapeutic treatments.
Another embodiment of the invention provides for methods for treating an animal by inhibiting the production of mucin in epithelial cells, particularly airway epithelial cells, by administering an effective amount of an inhibitor of mucomone-induced mucin production to the animal. Other methods, compounds and compositions are more fully described herein.